Assay instrumentation enables the interrogation of biological and chemical samples to identify components of the sample. The sample may be processed prior to performing the assay. The processed sample or assay may be placed in an assay tube and positioned in an internal compartment of a device for performing the assay and obtaining the results. The assay procedure may include the application of light, heat, enzymes, etc. The instrumentation includes computing and display components. The computing system controls the instrumentation and processing of gathered data. The display provides a graphical representation of the measured data. The cost and bulk of such instrumentation systems, such as medical diagnostic equipment or genetic testing equipment, commonly requires that biological samples be shipped to a testing facility for processing and analysis. This delays the receipt of test results, often by several days.
The nucleic acids DNA and RNA may be extracted from a biological sample in accordance with the Boom method or modifications thereof, for example, as is known in the art. In accordance with the Boom method, a biological sample is lysed and/or homogenized by mixing the biological sample with detergent in the presence of protein degrading enzymes. The chaotropic agents and silica or silica coated beads are mixed with the lysed biological sample. The chaotropic agents disrupt and denature the structure of nucleic acids by interfering with the macromolecular interactions mediated by non-covalent forces, such as hydrogen bonding, van der Waals forces, and hydrophobic interactions, for example. In the presence of the chaotropic agents, water is removed from the phosphate groups of the nucleic acids, exposing them and allowing hydrophobic bonding to the silica, such as silica or silica coated beads. Protein, cellular debris, and other substances in the biological samples do not bond to the silica and are retained in the solution. The silica beads are washed several times to remove non-nucleic acid materials, such as proteins, lipids, cellular constituents, including cellular molecules, and other substances found in biological samples. Silica coated magnetic beads may be used to assist in the separation of the nucleic acids bound to the silica coating from the solution, via a magnetic field or magnet. The nucleic acids are then eluted from the silica or silica coated beads into a buffer by decreasing the concentration of the chaotropic agents. The elution buffer may be pure water or Tris EDTA (“TE”) buffer, for example.
Polymerase chain reaction (“PCR”) is a biochemical process used in assay procedures to exponentially copy a target nucleic acid (DNA or RNA) sequence. The PCR process can be tailored to be highly specific and sensitive, allowing amplification of a low copy number sequence into a detectable quantity. The reaction requires a combination of a target nucleic acid sequence, a DNA polymerase, a primer (short DNA sequence that hybridizes to a target sequence complementary to the target DNA), deoxynucleotide triphosphates (“dNTPs”) (which are joined by the polymerase to the copied sequences), and a buffer solution including divalent cations (magnesium or manganese ions). The reaction proceeds in temperature cycles including: 1) a melting/denaturing stage during which the reaction mixture is brought to a relatively high temperature at which double stranded DNA separates into single strands; and 2) a lower annealing temperature, at which the primers attach to a complementary sequence and the polymerase join the dNPT to the 3′ end of the primer, forming a complimentary copy of the sequence. This copy can then act as a template for subsequent reaction cycles. Additional heating and cooling steps may be provided to optimize the process. The high sensitivity of PCR allows use in a diagnostic assay for detection of a pathogen without culturing, as may be required in alternative assays. The high sensitivity also reduces false negatives. The high specificity of PCR reduces false positives.
Quantitative Real-Time PCR (qPCR) is the real time detection of an amplified DNA or RNA sequence. This process can use intercalating dyes that fluoresce when exposed to an excitation wavelength after the dye binds to double stranded DNA. Alternatively, other chemistries are available, such as linear probes. Probe chemistries add another layer of specificity because specific hybridization between the probe and a target nucleic acid sequence is required to generate fluorescence.
One example of a linear probe is a hydrolysis probe, which are nucleic acid sequences that include a reporter dye, such as a fluorophore, on the 5′ end, and a fluorescent quenching moiety agent on the 3′ end. Such a probe generally relies on the 5′-3′ exonuclease activity of Taq Polymerase. The fluorescent quenching of the 5′ fluorophore requires that the quenching agent be in proximity of the 5′ fluorophore. The polymerase hydrolyzes the 5′ fluorophore during the extension phase of a PCR cycle, the fluorophore is removed from proximity to the quenching agent, allowing fluorescence from the dye to be detected. As in other real-time PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR.
Another probe chemistry that can be used are structured probes, such as molecular beacons. Molecular Beacons consist of a hairpin loop structure that is complementary to the target sequence and a stem complementary to the termini. One end of the termini contains a reporter dye and the other end contains a quencher dye which are brought in close proximity when the probe is in the hairpin state. Upon binding to its target the hairpin is opened and the fluorophore and quencher are separated, resulting in increased fluorescence. If the target sequence does not exactly match the Molecular Beacon sequence, hybridization and therefore fluorescence will not occur because the hairpin state is thermodynamically favored over the hybridized state.
qRT-PCR (Real Time quantitative Reverse Transcription PCR) enables reliable detection and measurement of RNA targets, such as mRNA and RNA viruses. An initial cycle of the reaction employs a reverse transcriptase to make a DNA copy from an RNA template. The copies of the DNA sequence are then amplified as with conventional PCR.
The functionality of personal electronic devices, such as smartphones and tablets, for example, is expanding. For example, smartphones are capable of wireless data transmission, global position tracking, image and video capture from front and rear facing cameras, data processing, data storage (including image storage), data display, time and date tracking, and acceleration measuring, for example.
Smartphones have been used in conjunction with medical devices for data collection and analysis. For example, AliveCor, Inc., San Francisco, Calif., provides an iPhone 4, 4S, and 5 case with a built-in heart monitor that enables performance of an electrocardiogram (ECG). The iPhone ECG can be used by consumers, for clinical diagnostics and in veterinary applications. iBGStar®, available from the Sanofi—Aventis Groupe, Frankfurt, Germany, provides a glucose meter for diabetics that plugs into the bottom of an iPhone. Mobisante, Inc., Redmond, Wash., has developed a handheld, smartphone-enabled ultrasound imaging device. CellScope, Inc., San Francisco, Calif., developed a smart-phone enabled otoscope for remote diagnoses of ear conditions, such as pediatric ear infections. Tinke, available from Zensorium, Singapore, monitors pulse, respiration, and blood oxygen levels. An iPhone App also displays pulse, respiration, and blood oxygen measurements, as well as composite score related to fitness and wellness of a user.